The present invention is directed to novel nucleic acid probes and protein probes. The invention is also directed to particular novel polyclonal and monoclonal antibodies useful for detecting the probes, and to novel hybridomas for making the monoclonal antibodies.
Nucleic acid probes, also called hybridization probes, allow specific polynucleotide sequences to be detected. Protein probes make possible detection of various compounds, such as proteins, steroids, carbohydrates, and nucleic acids that will undergo specific, usually noncovalent binding with the protein probe. When the protein probe is a labeled antibody, detection of a corresponding antigen is possible.
Nucleic acid probes can be used to detect specific polynucleotide sequences and can assist the diagnosis and treatment of numerous genetic disorders and diseases such as cystic fibrosis, muscular dystrophy, acquired immune deficiency syndrome (AIDS), hepatitis, herpes simplex, Epstein-Barr, and various viral infections such as those caused by cytomegalovirus and adenovirus.
Nucleic acid probes can also reveal genes coding antigens responsible for graft rejection. Genetic information useful in cancer oncogeny testing and forensic medicine can be obtained.
The hybridization probe or the protein probe can be made by labeling a polynucleotide or a protein with a detectable label in a modification or attaching reaction. Typically, the label is joined to a binding moiety before being attached to the polynucleotide or the protein. The binding moiety has a reactive group that functions to attach the label to the polynucleotide or to the protein. The label joined to the binding moiety is called a tag compound.
The hybridization probe can be contacted and incubated with another polynucleotide, called a capture polynucleotide, in a specific binding assay to determine if the two polynucleotides hybridize together. If the hybridization probe has been selected to include a polynucleotide, called the probe polynucleotide, which has at least one sequence or region substantially complementary to a sequence or region of the capture polynucleotide, then hybridization of the two polynucleotides occurs.
The protein probe can also be contacted and incubated with a specific binding partner in a specific binding reaction. Specific binding is generally strong but reversible, and does not normally lead to the formation or breaking of covalent bonds between or within members of the binding pair. Binding pairs can include antigen/antibody and receptor/receptor-binding molecule binding pairs.
Radioactive isotopes of atoms such as hydrogen (.sup.3 H), phosphorus (.sup.32 P), or iodine (.sup.125 I) are commonly used labels. Assays using radioactive labels require extensive safety precautions, expensive equipment and special waste treatment procedures often requiring a combination of federal, state, and local regulatory involvement. The instability of radioactive labels also results in high usage costs. Additionally, even with long exposure time, limited resolution is available in .sup.3 H and .sup.125 I autoradiography. Furthermore, the .sup.32 P isotope is a hazardous isotope. Hence, there is a need for nonradioactive labels for nucleic acid and protein probes.
In typical nonradioactive indirect labeling methodologies, a hapten is attached to a probe polynucleotide to serve as the indirect label. Alternately, the hapten can function as the indirect label by being attached to a protein. Following hybridization, the hapten-labeled probe polynucleotide can be contacted with an anti-hapten antibody, or a similar specific binding partner for the hapten. The anti-hapten antibody can be labeled with a detectable moiety such as, for example, an enzyme, chemiluminescent compound, or fluorescent compound. Alternately, a second antibody to the anti-hapten antibody can have the detectable moiety.
A difficulty in finding nonradioactive labels suitable for use in indirect labeling schemes is the requirement that such labels be haptenic, that is, capable of inducing an immune response when coupled to a carrier. The hapten label must also be capable of binding to its corresponding antibody. The hapten label also needs to be small enough not to interfere with the hybridization reaction, yet large enough to be "seen" by the anti-hapten antibody.
Biotin and its derivatives have been used as nonradioactive probe labels. Hybridized biotinylated probe is typically detected by contacting biotin label with avidin or an anti-biotin antibody. The biotin label is usually conjugated to a reactive group such as a hydrazide capable of attaching to or modifying a nucleotide. Prior-art biotin labels have included PHOTOBIOTIN.TM. (N-[4-azido-2-nitrophenyl]-N'-[N-d-biotinyl-3-aminopropyl]-N'-methyl-3-pro panediamine); biotin succinimide ester (biotin-NHS); and biotin hydrazide.
PHOTOBIOTIN.TM. can modify probe polynucleotide in about fifteen minutes, but is very expensive. PHOTOBIOTIN.TM. has the additional disadvantages of being unstable and extremely light sensitive, making the modification reaction difficult to control. Biotin hydrazide and biotin succinimide ester are not as expensive as PHOTOBIOTIN.TM., but both require much longer modification reaction times. Modification with biotin succinimide ester is a two-step procedure, while biotin hydrazide requires from twenty-four hours to approximately two-and-one-half days for completion of the modification reaction.
In addition to considerations of expense, stability, and modification reaction time, a "stickiness" or aggregation problem is encountered with biotin labels. Stickiness refers to the situation wherein biotinylated antibodies and/or nucleic acid probes aggregate due to the change in the surface characteristics of normally charged polynucleotides conjugated to neutral (uncharged) biotin. The aggregation problem is encountered with all biotin labels, regardless of the labeling conjugate used in preparation of the labeled probe. Aggregation results in reduced solubility and lower reactivity during the modification reaction.
Nonradioactive labels other than biotin have been sought for incorporation into probes to address these problems. The nonradioactive label can also be useful as an alternate nucleic acid and protein probe label. When biotin alternative labels are used in conjunction with biotinylated probes, sandwich assays can be performed in a single step.
Two nonradioactive, nonbiotin haptenic labels have recently met with some success. One is N-acetoxy-N-2-acetylaminofluorene (AAF) and its 7-iodo-derivative (AAIF). AAF and AAIF modify guanine residues. Another is methoxyamine (also referred to as o-methylhydroxylamine, methoxylamine, .alpha.-methylhydroxylamine, and hydroxylamine methyl ether). Commercial kits using methoxyamine-labeled probes are available from Sigma Chemical Company, St. Louis, Mo. (SulfoPROBE.TM.), FMC BioProducts, Rockland, Md. (CHEMIPROBE.TM.), and Orgenics, Ltd., Israel (CHEMIPROBE.TM.).
Neither of these two labels completely overcomes the problems associated with biotin labels. AAF is unstable. Methoxyamine is more stable but it is not very soluble, resulting in lower reactivity during the labeling process and less effective alleviation of the "stickiness" problem typically encountered with biotin labels. Both AAF and methyoxyamine are carcinogenic and must therefore be used with considerable caution.
What is needed therefore is a label for nucleic acids probes and protein probes that is: (1) haptenic; (2) stable; (3) reactive (for ease of labeling); (4) soluble (for both ease of labeling and to alleviate the "stickiness" problem); (5) noncarcinogenic; (6) nonradioactive; and (7) inexpensive.